Preliminary Characterization of Phospholipase A2 in Lagenidium giganteum

Joanna K. MacKichan, Amy R. Tuininga, James L. Kerwin

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3 Scopus citations

Abstract

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A2 in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A2 (PLA2) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA2 activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA2 activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1-14C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca2+, Mg2+, and Mn2+ all enhanced PLA2 activity, while Co2+, Fe2+, and Zn2+ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca2+ and Mn2+ than Mg2+, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA2 activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA2 hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA2 activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA2 activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.

Original languageEnglish
Pages (from-to)180-192
Number of pages13
JournalExperimental Mycology
Volume18
Issue number2
DOIs
StatePublished - 1 Jun 1994

Keywords

  • Lagenidium giganteum
  • Lipase
  • Oomycetes
  • cholesterol
  • oosporogenesis
  • phosphatidylcholine
  • phospholipase A
  • phospholipids

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